PROJECT SUMMARY Autophagy is a complex catabolic process that degrades cytosolic components within the lysosome and plays a prominent role in maintaining cellular homeostasis. Although widely implicated in the pathophysiology of numerous diseases, the detailed regulation of the separate steps of the autophagy process remains underexplored. To gain insight into this important question, we propose to characterize a novel autophagy- regulatory pathway that we recently identified, involving the conserved mammalian Hippo kinases STK3/STK4 and the essential Atg8 autophagy protein family. We have reported that STK3 and STK4, the mammalian orthologs of Hippo kinase, are essential for autophagy in diverse organisms, and both can phosphorylate the Atg8 family member LC3B on amino acid Thr50. STK3/4-mediated phosphorylation is critical for fusion of autophagosomes with lysosomes, a late step in the autophagy process, as well as the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Our discovery of this novel mode of autophagy regulation involving direct phosphorylation of LC3B by STK3/4 provide a unique opportunity to gain new mechanistic insight into the regulation of the autophagy process, including the Atg8 family of proteins. To this end, we will address the molecular and cellular circuitry of autophagy regulation mediated by STK3/STK4 by using a strong combination of proteomic, molecular and cytological techniques. Specifically, we will use biochemical approaches to characterize STK3/4 interactions with other members of the Atg8 family, besides LC3B, since STK3/4 bind additional Atg8 proteins yet it is not clear if they also can serve as substrates. In Aim 2, we will use cytological and proteomic approaches to dissect the intracellular- and molecular consequences of phosphorylation of Atg8 proteins by STK3/4. Finally, in Aim 3, we will devise targeted RNAi screens in conjunction with STK4 proteomic studies to identify regulators critical for the novel STK3/4-LC3B/Atg8 axis to regulate autophagy. These studies are innovative because we will use cutting-edge techniques to elucidate the molecular mechanism by which the Hippo kinases STK3/4 regulate autophagy via the key autophagy proteins in the Atg8 family. Moreover, these studies are likely to have significant impact because they will provide important information on how STK3/4 kinases and the Atg8 family regulate autophagy in mammals; such insights could prove important not only to our basic understanding of the autophagy process, but may also help develop future treatments against autophagy-linked diseases.